Parkinson's disease (PD) is a neurodegene Parkinson's disease (PD) is a neurodegenerative disorder occurring after an extensive loss of midbrain dopamine (DA) neurons that regulate normal body movements. The small protein, [unreadable]D-synuclein (a-Syn), is implicated in PD by its abundance in Lewy bodies and by geo 14-3-3 chaperones to regulate the activity of key enzymes. One such enzyme is tyrosine hydroxylase (TH), which is rate limiting for DA synthesis, and a protein that can be activated by 14-3-3 binding. Using in vitro models our laboratory discovered that a-Syn binds to and inhibits TH. We also found that a-Syn also inhibits DA synthesis by reducing the activity of the second enzyme in the DA biosynthetic pathway, aromatic amino acid decarboxylase (AADC). Short term regulation of TH and AADC activity is mediated by changes in protein phosphorylation/dephosphorylation. TH is known to be dephosphorylated by the protein phosphatase PP2A. Recent findings from our laboratory reveal that a-Syn binds to and activates this very phosphatase. Cumulatively, the data lead us to hypothesize that soluble a-Syn contributes to normal DA levels by inhibiting TH and AADC, but as a-Syn aggregates DA is overproduced, initiating a feed-forward cascade of reactive species that compromises nigrostriatal dopaminergic neuronal viability. The goal of this proposal is to extend and confirm our in vitro findings in vivo using mouse models to measure the impact of a-Syn on PP2A and TH with regard to DA synthesis. To achieve this, we will use multiple transgenic lines of a-Syn mice and assays utilizing recombinant proteins. In vivo we will modulate a- Syn levels by lentiviral transduction and then measure the effects of soluble and aggregated a-Syn on TH and PP2A by immunoblots, enzyme assays, immunohistochemistry, and confocal microscopy. In vitro we will use recombinant proteins to measure the impact of aggregated a-Syn on TH and PP2A activity as measured using well characterized enzymatic assays as we test the following hypotheses: 1) that a-Syn inhibits DA synthesis by effects on TH and PP2A in vivo, and 2) that soluble a-Syn stimulates PP2A and inhibits TH activity, while aggregated a-Syn is unable to do so, as measured in vitro and in vivo. We are uniquely positioned to elucidate the biological and translational relevance of a-Syn as a regulator of DA homeostasis, with the potential to identify novel preventative or restorative PD therapies.